Primer Design and Optimisation of Pongo pygmaeus CFTR Gene: Eight High Mutation Exon (Range No. 1-11)

Abstract

Orangutans are known to have respiratory diseases similar to cystic fibrosis symptoms in humans. Cystic fibrosis is a respiratory disease caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR). Research on the CFTR gene in Bornean orangutans is still rarely done. Therefore, this study will detect the CFTR gene mutation of the Bornean orangutan samples from the BOSF rehabilitation centre. This study aimed to design specific primers for the CFTR gene in several predefined gene exons with the densest mutation and to obtain the optimal annealing temperature for each primer. We used UGENE version 40.1 for manual primers design. Optimisation of PCR amplification program and primer annealing temperature was determined with pre-denaturation at 94 °C, denaturation at 95 °C, different annealing temperatures for each primer, and final extension at 72 °C. The optimum condition for annealing temperatures of designed specific primers for exons 3, 4, 6, 8, and 11 of the orangutan CFTR gene was at 51-58 °C. Primer can amplify the exon and intron regions flanking the target exon.